CN110734503A

CN110734503A - Arab galactooligosaccharides, and preparation and application thereof

Info

Publication number: CN110734503A
Application number: CN201911152186.1A
Authority: CN
Inventors: 贺亮; 施锴云; 程俊文; 王衍彬; 王进; 魏海龙; 胡传久; 李海波
Original assignee: Zhejiang Academy of Forestry
Current assignee: Zhejiang Academy of Forestry
Priority date: 2019-11-22
Filing date: 2019-11-22
Publication date: 2020-01-31
Anticipated expiration: 2039-11-22
Also published as: CN110734503B

Abstract

The invention discloses arabino-galactooligosaccharides and preparation and application thereof, wherein the arabino-galactooligosaccharides comprise polysaccharides with weight percentage of more than 99%, the polysaccharides comprise arabinose and galactose, the molar ratio of the arabinose to the galactose is 1:16, the weight average molecular weight of the arabino-galactooligosaccharides is 4000Da-5000Da, the preparation method adopts two-step enzymolysis, and adopts an ultrafiltration and nanofiltration coupling mode to separate and purify the degraded arabino-galactooligosaccharides, so as to directionally obtain the arabino-galactooligosaccharides with the weight average molecular weight of 4000Da-5000Da, thereby greatly shortening the purification time and solving the problem that the prior art is not easy to separate the molecular weight oligosaccharides with a certain fixed value.

Description

Arab galactooligosaccharides, and preparation and application thereof

Technical Field

The invention relates to the technical field of polysaccharides, in particular to arabinogalactan oligomers and preparation and application thereof.

Background

Arabinogalactan (AG) is -class neutral polysaccharide with high branched chain, and has multiple biological activities such as anti-tumor, anti-oxidation, immunoregulation, etc., it has been shown that AG also has the function of regulating intestinal tract, and has been approved as food additive by FDA certification in USA in 2002⁵Da, the structure of which mainly comprises galactan as a main chain, arabinose as a branch chain thereof and galactose are connected through β -1,3 bonds or β -1,6 bonds, but the relative molecular mass of Da is large, and the Da needs to be degraded to improve the biological activity of Da.

However, the degradation can not directionally obtain the oligosaccharide with the required molecular weight, and experiments show that the degraded arabino-galacto-oligosaccharides are discontinuously distributed and span 1 × 10³To 5X 10³In addition, small molecular saccharides such as glucose, lactose and the like can be contained in degradation products in different degrees, and the distribution makes it more difficult to separate polysaccharide with single molecular weight, so that the subsequent physicochemical research and structural analysis of the polysaccharide are influenced, therefore, the directional separation of the oligosaccharide needs to be carried out according to different requirements, the directional separation preparation of the arabino-galacto-oligosaccharide by using a membrane separation method is not yet reported at presentChinese patent application CN 102676609B discloses method for efficiently preparing type uneven marasmius androsaceus polysaccharide by using membrane separation technology, which sequentially adopts inorganic ceramic microfiltration membrane and 4000-9000k inorganic ceramic ultrafiltration membrane to concentrate and purify to obtain uneven type marasmius androsaceus polysaccharide, and adopts spray drying method to prepare polysaccharide solution into powder product, the method has low preparation cost, the prepared product has good quality and long shelf life.

The existing degradation method of polysaccharide can not control the position of the broken glycosidic bond, which brings the problem that the molecular weight distribution of the degraded polysaccharide is wide, so that the degraded product can simultaneously contain the polysaccharide with target molecular weight, polysaccharide and oligosaccharide with small molecular weight and polysaccharide with large molecular weight, which are generated by excessive degradation, and therefore methods for obtaining the functional oligosaccharide polysaccharide in an oriented way need to be established.

In addition, most of the existing reports aiming at the polysaccharide relate to -level structural research of the polysaccharide, the analysis of the high-level structure of the polysaccharide is less, for example, the research on the monosaccharide composition, the glycosidic bond connection mode and the like of the polysaccharide is less, more and more researches show that the important functions of the polysaccharide are determined by the structural characteristics of the polysaccharide, the high-level structures (secondary and tertiary structures) are more compact, the biological activity of the polysaccharide is closely related to the molecular weight and the molecular chain conformation of the polysaccharide, and the understanding of the molecular weight, the molecular chain conformation and the like of the polysaccharide molecules is more helpful to clarify the biological activity action mechanism of the polysaccharide.

Disclosure of Invention

The invention aims to provide arabinogalactans with the weight-average molecular weight of 4000Da-5000Da, which have biological activity and obviously enhanced activity compared with arabinogalactan.

Another purpose of the invention is to provide the preparation method of the arabino-galacto-oligosaccharide, the method adopts enzyme degradation and membrane method separation orientation to obtain the bioactive functional oligosaccharide with the weight average molecular weight of 4000Da-5000Da, and the method has the advantages of simple operation and easy control, and is suitable for industrial large-scale production.

The invention also provides application of the arabinogalactan , which has a strong oxygen free radical scavenging effect, can be used as an antioxidant and also can be used for preparing the antioxidant, and has an in-vitro growth promoting effect on probiotics and can be used as a carbon source for promoting the proliferation of the probiotics.

The technical scheme adopted by the invention for solving the technical problems is as follows:

arabinooligosaccharides consisting of more than 99 wt.% of polysaccharides consisting of arabinose (Araf) and galactose (Galp) in a molar ratio of arabinose to galactose of 1:16, said arabinooligosaccharides having a weight average molecular weight of 4000Da to 5000Da (preferably 4500 Da).

preferably, the arabinose is α -arabinose, preferably α -L-arabinose, and the galactose is β -galactose, preferably β -D-galactose.

The building blocks of the polysaccharide preferably have (1 → 3) linked β -D-galactose (β -D-Galp) residues (i.e., 1,3 glycosidically linked β 1-D-galactose residues) as the backbone, substituted at the 6-positions of two β 2-D-galactose residues adjacent to the backbone by a branched chain β 0 and a terminal α -arabinose (α -Araf), respectively, ( further preferably α -L-arabinose (α -L-Araf)), and branched chains are (1 → 6) linked β 3-D-galactose ((1,6) - β 4-D-Galp) residues and a terminal β -D-galactose (β -D-Galp).

The preparation method of the arabinogalactan comprises the following steps:

(1) degrading, namely uniformly mixing 0.05mol/L citric acid buffer solution with β -1, 3-galactose incision enzyme and arabinogalactan, adjusting the pH value, carrying out enzymolysis for times, removing precipitates to obtain supernatant (namely partially degraded arabinogalactan degradation solution), continuously adding α -arabinosidase into the supernatant, adjusting the pH value, carrying out secondary enzymolysis, and removing the precipitates to obtain the degradation solution;

(2) and (3) membrane separation and purification: sequentially adopting an ultrafiltration membrane and a nanofiltration membrane to carry out membrane separation and purification on the degradation liquid obtained in the step (1), wherein the collected ultrafiltration membrane permeate is separated and purified by the nanofiltration membrane, and the collected nanofiltration membrane retentate is collected to obtain a separation liquid;

(3) and (3) drying: concentrating and drying the separated liquid obtained in the step (2) to obtain the arabinogalactan, which is named as Oligo-AG 2.

In the invention, the permeation liquid flows out from the permeation port of the outer membrane cavity through the permeation membrane, and the trapped liquid flows back to the sample tank from the reflux port without permeating through the membrane.

In order to achieve better effects of the invention, it is preferable that:

in the step (1), the arabinogalactan can be a commercially available product or prepared by adopting the existing preparation method.

The dosage relation of the β -1, 3-galactose incision enzyme and the arabinogalactan is 10U-45U: 1g, which is beneficial to obtaining a target product.

The usage amount of the α -arabinosidase and the arabinogalactan is 20U-65U: 1g, the optimal usage amount is 40U-65U: 1g in the step , and the target product is obtained.

The pH value of times of enzymolysis is preferably 4.5-7.0 (preferably 5.0-7.0 in the step of ), the enzymolysis temperature is preferably 35-55 ℃ (preferably 45-55 ℃ in the step of ), and the enzymolysis time is preferably 30-60 h.

The pH value of the secondary enzymolysis is preferably 5.5-7.5, the enzymolysis temperature is preferably 45-65 ℃ ( step is preferably 50-65 ℃), and the enzymolysis time is preferably 30-60 h.

The ratio of the volume of the citric acid buffer solution to the weight of the arabinogalactan is preferably (20mL-200 mL): 0.1g-1g), and further is preferably (20mL-40 mL): 0.1g-0.8 g.

In the step (2), the molecular weight cut-off of the ultrafiltration membrane is 3000Da-10000Da, and the step is preferably 4000Da-8000 Da.

The molecular weight cut-off of the nanofiltration membrane is 200Da to 1000Da, and the molecular weight cut-off is preferably 200Da to 800Da in the step .

The parameters of membrane separation and purification by adopting ultrafiltration membrane and nanofiltration membrane are that the pressure difference is 0.1MPa-0.5MPa (0.15 MPa-0.3MPa is preferable in step ), the temperature of degradation liquid is 15 ℃ -50 ℃ (20 ℃ -30 ℃ is preferable in step ), and the membrane area is 1.5m²-5m²(step is preferably 1.5m²-2.5m²) Flux of degraded liquid film is 0.1m²/h-2.5m²H (step is preferably 0.2 m)²/h-0.8m²H). The parameters of the ultrafiltration membrane and the nanofiltration membrane for membrane separation and purification can be the same or different.

The ultrafiltration membrane and the nanofiltration membrane are subjected to membrane separation and purification by adopting a tangential flow mode; high separation efficiency and good purification effect.

The degradation liquid is sequentially subjected to membrane separation and purification steps by adopting an ultrafiltration membrane and a nanofiltration membrane, preferably, the degradation liquid is firstly diluted, the separation effect is better, and the specific steps comprise that the degradation liquid is diluted by water until the weight percentage of the degradation liquid is 40% -80% ( is preferably 50% -70%).

The arabinogalactan has biological activity, is obviously enhanced compared with arabinogalactan, has an in-vitro growth promotion effect on probiotics, can be used as a carbon source to promote the proliferation of the probiotics, can be used as a probiotic growth promoter or used for preparing the probiotic growth promoter, and can be used as functional oligosaccharide to be applied to the fields of food additives, health products and medicines, wherein species of bifidobacteria and clostridium butyricum are preferably selected as the probiotics.

The arabinogalactan has the capability of scavenging oxygen free radicals, and the OH free radical clearance of the arabinogalactan is far greater than that of arabinogalactan under the premise of the same concentration in the OH free radical clearance measurement; the arabinogalactan has stronger anti-oxygen free radical activity, can be directly used as an antioxidant or used for preparing the antioxidant, and can be used in the fields of food additives, health care products and medicines.

Compared with the prior art, the invention has the following advantages:

the weight average molecular weight of the arabino-galacto-oligosaccharide can be fixed to 4000Da-5000Da, particularly 4500Da, the monosaccharide composition of the polysaccharide part of the arabino-galacto-oligosaccharide is arabinose and galactose, and the molar ratio of the arabinose to the galactose is 1:16, so that technical support is provided for the practical application of natural polysaccharide products, and the arabino-galacto-oligosaccharide has -wide application prospect.

Compared with arabinogalactan, the molecular weight of the oligosaccharide after enzyme degradation is reduced, the branched chain substitution degree of the arabinogalactan, namely the number ratio of arabinose to galactose, is relatively reduced, the biological function activity of the arabinogalactan is obviously enhanced, and the oligosaccharide with a simple structure can be used as a microbial carbon source to enable the microbial fermentation to be quicker, so that the method not only can support the increase of the abundance of probiotics such as bifidobacterium and the like in the fermentation process, but also can increase short-chain fatty acids generated by the probiotics and has an obvious in-vitro growth promotion effect on the probiotics such as bifidobacterium and the like, and therefore, the arabinogalactan can be used as a functional oligosaccharide to be applied to the fields of food additives, health care products and medicines.

The preparation method provided by the invention has the advantages that the arabinogalactan is degraded in two steps by adopting β -1, 3-galactose incision enzyme and α -arabinosidase in a mild environment, the degraded arabinogalactan is separated and purified by adopting an ultrafiltration and nanofiltration coupling mode, the arabinogalactan with the weight average molecular weight of 4000Da-5000Da (preferably 4500Da) is obtained in an oriented mode, the purification time is greatly shortened, the arabinogalactan can be degraded in an oriented mode to a fixed value molecular weight, the prebiotic activity of the oligomeric polysaccharide with the molecular weight in the section is obviously enhanced, and the problem that the oligomeric polysaccharide with the molecular weight of is not easy to separate in the prior art is solved.

The invention has no phase change in the whole process of separating and purifying the arabino-galacto-oligosaccharide by using a membrane method, can effectively control the molecular weight of a degradation product, namely the arabino-galacto-oligosaccharide, can effectively remove micromolecules in a degradation solution, greatly improves the yield of a target product, is green and environment-friendly, and cannot pollute the environment.

Drawings

FIG. 1 is a graph showing the results of the absolute weight-average molecular mass of degraded arabinogalactans in example 4, wherein the abscissa represents the time to peak (time/min) and the ordinate represents the relative value of the response of the instrument (relative scale).

FIG. 2 is the IR spectrum of example 5, wherein FIG. 2(a) shows AG at 4000-400cm^-1An infrared spectral scan over a range of wavelengths; FIG. 2(b) shows Oligo-AG2 at 4000-400cm^-1Infrared spectral scan over a range of wavelengths.

FIG. 3 is a nuclear magnetic resonance spectrum of Oligo-AG2 of example 6, wherein FIG. 3(a) is that of Oligo-AG2¹H-NMR spectrum, FIG. 3(b) of Oligo-AG2¹³C-NMR spectrum, FIG. 3(C) is HMBC spectrum of Oligo-AG2, and FIG. 3(d) is COSY spectrum of Oligo-AG 2.

FIG. 4 shows the structural formula of Oligo-AG2 resolved by structural analysis in example 6.

FIG. 5(a) is an atomic force microscopy scan of AG in example 7, and FIG. 5(b) is an atomic force microscopy scan of Oligo-AG2 in example 7.

FIG. 6(a) is a graph showing the effect of arabinogalactan and arabinogalactooligosaccharide on the growth curve of Bifidobacterium animalis in example 8.

FIG. 6(b) is a graph showing the effect of arabinogalactan and arabinogalacto-oligosaccharide on the growth curve of Clostridium butyricum in example 8.

Detailed Description

The invention is further illustrated in below in conjunction with the attached drawings and the detailed description, it being understood that these examples are given by way of illustration only and are not intended to limit the scope of the invention.

Example 1

Preparation of arabino-galacto-oligosaccharides:

(1) and (2) degradation, namely, taking 0.1g of arabinogalactan to a 100mL hydrolysis bottle, adding 20mL of 0.05mol/L citric acid buffer solution into the hydrolysis bottle, adding β -1, 3-galactose incision enzyme according to the dosage of 10U/g of arabinogalactan (namely, adding 10U β -1, 3-galactose incision enzyme into each gram of arabinogalactan), adding distilled water to ensure that the total volume of enzymolysis is 50mL, adjusting the pH value to be 5.0, carrying out enzymolysis for 30h in a constant-temperature shaking table at the temperature of 200 r/min and 45 ℃, centrifuging to remove precipitates to obtain a supernatant, continuously adding α -arabinosidase according to the dosage of 65U/g of arabinogalactan into the supernatant (namely, adding 65U α -arabinosidase into each gram of arabinogalactan), adding distilled water to ensure that the total volume of enzymolysis is 50mL, adjusting the pH value to be 5.5, carrying out enzymolysis for 30h in a constant-temperature shaking table at the temperature of 200 r/min and 50 ℃, centrifuging to remove precipitates, and finally obtaining a degraded supernatant, namely, the degraded supernatant.

(2) And (3) membrane separation and purification: diluting the degradation liquid obtained in the step (1) with distilled water until the weight percentage of the degradation liquid is 50%, sequentially adopting an ultrafiltration membrane with the molecular weight of 4500Da and a nanofiltration membrane with the molecular weight of 200Da for membrane separation and purification, wherein the membrane areas are 1.5m²The membrane flux was 0.2m²And h, performing membrane separation in a tangential flow mode under the conditions that the pressure difference is 0.15MPa and the temperature of the feed liquid is 20 ℃, wherein the collected ultrafiltration permeating liquid is separated and purified by a nanofiltration membrane, and the nanofiltration trapped liquid is collected to obtain a separated liquid.

(3) And (3) freeze drying: and (3) evaporating, concentrating and freeze-drying the separated liquid obtained in the step (2) to obtain purified arabinogalactan, which is named as Oligo-AG 2.

The weight average molecular weight of Oligo-AG2 was determined to be 4500 Da.

Example 2

Preparation of arabino-galacto-oligosaccharides:

(1) and (2) degradation, namely, taking 0.5g of arabinogalactan to a 100mL hydrolysis bottle, adding 30mL of 0.05mol/L citric acid buffer solution into the hydrolysis bottle, adding β -1, 3-galactose incision enzyme according to the dosage of 45U/g of arabinogalactan (namely, adding 45U β -1, 3-galactose incision enzyme into each gram of arabinogalactan), adding distilled water to ensure that the total volume of enzymolysis is 50mL, adjusting the pH value to be 7.0, carrying out enzymolysis for 60h in a constant-temperature shaking table at the temperature of 200 r/min and 55 ℃, carrying out centrifugation to remove precipitates to obtain a supernatant, continuously adding α -arabinosidase according to the dosage of 50U/g of arabinogalactan into the supernatant (namely, adding 50U α -arabinosidase into each gram of arabinogalactan), adding distilled water to ensure that the total volume of enzymolysis is 50mL, adjusting the pH value to be 7.5, carrying out enzymolysis for 60h in a constant-temperature shaking table at the temperature of 200 r/min and 65 ℃, carrying out centrifugation to remove precipitates to obtain a degraded supernatant, namely, and finally obtaining the degraded supernatant.

(2) And (3) membrane separation and purification: diluting the degradation liquid obtained in the step (1) with distilled water until the weight percentage of the degradation liquid is 60%, sequentially adopting an ultrafiltration membrane with the molecular weight of 5000Da and a nanofiltration membrane with the molecular weight of 500Da for membrane separation and purification, wherein the membrane areas are both 2.0m²The membrane flux was 0.5m²And h, performing membrane separation in a tangential flow mode under the conditions that the pressure difference is 0.20MPa and the temperature of the feed liquid is 25 ℃, wherein the collected ultrafiltration permeating liquid is separated and purified by a nanofiltration membrane, and the nanofiltration trapped liquid is collected to obtain a separated liquid.

The weight average molecular weight of Oligo-AG2 was determined to be 4500 Da.

Example 3

Preparation of arabino-galacto-oligosaccharides:

(1) and (2) degradation, namely, taking 0.8g of arabinogalactan to a 100mL hydrolysis bottle, adding 40mL of 0.05mol/L citric acid buffer solution into the hydrolysis bottle, adding β -1, 3-galactose incision enzyme according to the dosage of 30U/g of arabinogalactan (namely, adding 30U β -1, 3-galactose incision enzyme into each gram of arabinogalactan), adding distilled water to ensure that the total volume of enzymolysis is 50mL, adjusting the pH value to be 5.5, carrying out enzymolysis for 48h in a constant-temperature shaking table at the temperature of 200 r/min and 45 ℃, centrifuging to remove precipitates to obtain a supernatant, continuously adding α -arabinosidase according to the dosage of 40U/g of arabinogalactan into each gram of arabinogalactan (namely, adding 40U α -arabinosidase into each gram of arabinogalactan), adding distilled water to ensure that the total volume of enzymolysis is 50mL, adjusting the pH value to be 6.5, carrying out enzymolysis for 50h in a constant-temperature shaking table at the temperature of 200 r/min and 50 ℃, centrifuging to remove precipitates to obtain a degraded supernatant, namely, the degraded supernatant.

(2) And (3) membrane separation and purification: diluting the degradation liquid obtained in the step (1) by using distilled water to the weight percentage of the degradation liquidThe ratio is 70%, sequentially adopting ultrafiltration membrane with molecular weight of 8000Da and nanofiltration membrane with molecular weight of 800Da for membrane separation and purification, wherein the membrane area is 2.5m²The membrane flux was 0.8m²And h, performing membrane separation in a tangential flow mode under the conditions that the pressure difference is 0.30MPa and the temperature of the feed liquid is 30 ℃, wherein the collected ultrafiltration permeating liquid is separated and purified by a nanofiltration membrane, and the nanofiltration trapped liquid is collected to obtain a separated liquid.

The weight average molecular weight of Oligo-AG2 was determined to be 4500 Da.

The following are examples of structural identification or performance analysis of Oligo-AG 2:

example 4: molecular weight detection

The specific test flow is as follows: the sample volume is 200-300 μ L (i.e. 2-3 times the sample ring volume), data is collected for 60min, and the column temperature is room temperature. Preparing 0.1mol/L NaNO₃Aqueous solution (containing NaN with mass percent concentration of 0.02 percent)₃) As mobile phase, pass through 0.22 μm membrane, ultrasonic degassing for 30 min. 3mg of the sample was weighed and dissolved in 1mL of 0.1mol/L NaNO₃Aqueous solution (containing NaN with mass percent concentration of 0.02 percent)₃) In the process, the mixture is dissolved for 4 hours by magnetic stirring and then passes through a 0.22 mu m film. The results of the absolute weight average molecular weight of the degraded polysaccharide using the arabino-galacto-oligosaccharides of example 1 are shown in FIG. 1.

FIG. 1 is a result chart of absolute weight average molecular weight of the separation effect of the oligomeric polysaccharide by enzymatic degradation and membrane separation, and it is known from FIG. 1 that the oligomeric polysaccharide prepared by enzymatic degradation and membrane separation can directionally obtain the target molecular weight, thereby greatly reducing the separation and purification time. And the activity of arabinogalactan having an absolute weight average molecular mass (Mw) of 4500Da, as determined by light scattering, was significantly increased.

Example 5: infrared spectroscopic analysis

AG2.0mg and Oligo-AG22.0 mg in the example were put in an agate mortar together with 200mg of KBr pellets, respectively, and ground into powder under irradiation of infrared light. The mixture is mixed evenly and the appropriate amount of the mixture is made into transparent tablets by a tabletting method. And carrying out preliminary analysis on the configurations of the two polysaccharides before and after degradation.

Infrared spectroscopic analysis of AG with Oligo-AG2 in the examples was as follows:

as shown in FIGS. 2(a) and 2(b), the polysaccharide AG and Oligo-AG2 were at 4000-400cm^-1The distinct characteristic absorption peaks of the polysaccharide are present in the range, and the peak types are quite similar, which indicates that the enzymatic degradation of the invention does not change the backbone structure of the polysaccharide. The two polysaccharides are near 3400cm^-1At and near 2930cm^-1Each has wide characteristic peaks of O-H stretching vibration and relatively weak peaks of C-H stretching vibration at 1600cm^-1、1400cm^-1Strong absorption peaks existing near the sugar molecule are respectively C-H angle change characteristic peaks and bending vibration characteristic peaks of the sugar molecule; at 1200-^-1And C ═ O as a stretching vibration absorption peak between the two peaks. The polysaccharides are all at 836-884cm^-1There are absorption peaks between them, which indicate that the polysaccharide mainly contains α, β -glycosidic bonds, the infrared spectrum of Oligo-AG2 in examples 1-3 is 4000-400cm^-1The characteristic absorption peak and peak shape of the polysaccharide in the range are the same.

Example 6: nuclear magnetic resonance spectroscopy

60mg of the lyophilized Oligo-AG2 sample of the example was dissolved in 1mLD₂Centrifuging, removing precipitate, vacuum freeze drying, and dissolving the freeze dried sample in 1mLD₂Performing O neutralization, centrifuging, removing precipitates, performing vacuum freeze drying, and repeatedly treating for 4 times; finally, the sample after repeated freeze-drying is used for 1mL of D₂Dissolving O in 5mm nuclear magnetic tube, scanning with Bruker AVANCE 600 nuclear magnetic resonance apparatus in Switzerland at 600MHz to obtain hydrogen nuclear magnetic resonance spectrum¹H-NMR spectrum), carbon spectrum: (¹³C-NMR spectrum), TOCSY spectrum, HMBC spectrum, etc.

NMR analysis of AG with Oligo-AG2 in the examples was as follows:

chemical shifts of the residues are shown in Table 1, analysis deduces chemical shifts of hydrogen and carbon of all residues, coupling constants are compared with standard residues to find that D is assigned to Ara sugar residue, A, B, C is assigned to Gal sugar residue, can judge the configuration of anomeric carbon by the chemical shift of anomeric hydrogen of each sugar residue, delta >5.00ppm is α -type, delta <5.00ppm is β -type, and the chemical shifts of Ara anomeric hydrogen are relatively low field (delta >5.00), α -configuration and β -configuration from nuclear magnetic analysis results.

TABLE 1 chemical shift assignments (δ, ppm) for Oligo-AG2 saccharide residues

A signal of approximately 106.0 is assigned to A (1,3,6- β -Galp), B (1,3- β -Galp) and C (1,6- β -Galp), both residues A and C containing a C-6 bond, but residue B containing a C-3 bond, thus, residue B is 1,3- β -Galp, residue C is 1,6- β -Galp, residue A is 1,3,6- β -Galp, the resonance at 112.09 corresponds to terminal D, and for residue D (T-L-Araf), a match of C-1 signal 112.09 and C-5 of Ara at 77.73 indicates the presence of terminal α -Araf.

The and two-dimensional nuclear magnetic spectra of Oligo-AG2 are shown in FIG. 3 (a-D). Oligo-AG2 in examples 1, 2 or 3 is neutral AG with 1,3,6-, 1, 3-and 1, 6-linked galactosyl backbone in molar ratio 1:6: 1. it was found by study that Oligo-AG2 has novel structural features in that it is structured with 1,3 glycosidically linked -galactose residues as backbone and 1,3 glycosidically linked 6 positions substituted with (1,6) - -D-Galp or terminal Araf as backbone, as shown in FIG. 4, in particular, -D-galactose ( 7-D-Galp) residues linked with (1 → 3) (i.e.5968-D-galactose residues linked β -D-galactose residues) as backbone and two β -D-galactose residues adjacent 636 positions are substituted with branched chains of (3-D-galactose residues) with (3-7-D-Galp) residues (i.e.3-D-galactose residues) as backbone, (i.5968-D-galactose residues linked) as backbone, (i.3-3-7-D-Galp) residues are substituted with a molar ratio of (11-3-arabinose residues; preferably, 3-7-D-3-7-D-galactose residues as side chains) as opposed to terminal chains as side chains, (3-19, 3-7-19, 3-7-3-.

Example 7: atomic Force Microscope (AFM) topography analysis

(1) Preparation of sample solution: 1mg of dried AG and the Oligo-AG2 sample in the example were weighed, dissolved in 1mL of SDS (sodium dodecyl sulfate, concentration 0.1mg/mL) aqueous solution, stirred in water bath for 2h, heated to promote complete dissolution of the sample, cooled to room temperature, diluted with 0.1mg/mL SDS aqueous solution step by step to a sample concentration of 50. mu.g/mL, and dissolved by magnetic stirring for 24 h.

(2) Preparation of AFM samples: 2 mmol. mu.L of 5. mu.L^-1Silicon dichloride (NiCl)₂) The aqueous solution was placed on freshly peeled mica flakes. Adsorbing for 10min, washing with ultrapure water, and naturally drying in air; mu.L of AG and 5. mu.L of Oligo-AG2 solution were dropped on the surface of the mica sheet, left in the air for 10min, and then a large amount of ultrapure water was absorbed by a syringe to wash away the unadsorbed residue. Dried for use (all reagents required 0.22 μm filters).

(3) Conditions for observation of AFM samples: the sample observation was performed under the conditions of room temperature and air humidity of 50% -60%, and the apparatus model was XE-70 atomic force microscope, a Korean Park company. Probe Si₃N₄Has a microcantilever length of 200 μm and a force spring constant of 0.2N/m, and images were obtained in the tapping mode.

AFM topography analysis of AG and Oligo-AG2 was as follows:

FIG. 5(a) is AFM map of AG, FIG. 5(b) is AFM map of Oligo-AG2, sample concentration is 50 μ g/mL, AFM is observed that AG and Oligo-AG2 are in a large amount of molecular chains with random coil shape in aqueous solution, which are flatly spread on mica sheet and have many branches to form random coil conformation, furthermore, -like single-chain polysaccharide molecules have diameter ranging from 0.1 to 1.0nm, in this study, original AG is measured to be in a flexible chain shape with 1.32nm of unit girth molar mass ML being 782nm^-1The length q of the continuation was 5.43nm, and d was 1.05 nm. The degraded Oligo-AG2 molecule is smaller, the diameter of the molecule becomes 0.78nm, but still presents short and flexible chain shape.

Example 8: evaluation of biological Activity of AG and Oligo-AG2

The oligosaccharide Oligo-AG2 obtained in example 1 was used to determine the growth promoting effect of polysaccharide on two probiotics before and after degradation in an in vitro model.

The method comprises the following specific steps:

activating strains: under the condition of aseptic and anaerobic operation, dissolving Bifidobacterium animalis and Clostridium butyricum lyophilized powder in a basal culture medium, inoculating, performing anaerobic culture at 37 +/-1 ℃ for 48h, and continuously performing subculture according to the inoculum size of 5 percent (by weight). After activation, passage is carried out for 3 times, and the 3 rd generation bacterium liquid is taken and stored at the temperature of 80 ℃ for later use.

In vitro culture: 50mL of improved MRS culture medium (provided by Qingdao Haibo biotechnology, Inc.) is added into a 150mL triangular flask, 3 rd generation bacterial liquid of animal bifidobacterium and 3 rd generation bacterial liquid of clostridium butyricum are respectively added, polysaccharide with the mass fraction of 0.5 percent is added, standing culture is carried out for 48 hours, 0.1mL of fermentation liquid is absorbed from the triangular flask every 4 hours, a flat plate counting method is adopted to determine the number of viable bacteria in the culture medium, and the influence of the arabino-galactooligosaccharide on the external growth curve of the two probiotics is researched by taking a glucose base culture medium as a reference.

From fig. 6(a) and fig. 6(b), it can be seen that, within , the log of viable count of bifidobacterium in the culture medium with AG as carbon source is 11.20% higher than that of the control group, and the log of viable count of clostridium butyricum is 13.23% higher than that of the control group, while, within , the log of viable count of bifidobacterium in the culture medium with Oligo-AG2 is 14.34% higher than that of AG, and the log of viable count of clostridium butyricum is 13.28% higher than that of AG, compared with AG, within , the log of viable count of bifidobacterium in the culture medium with Oligo-AG2 in examples 2 and 3 is 14.32% and 14.35% higher than that of AG, the log of viable count of clostridium butyricum is 13.20% and 13.25% higher than that of AG, compared with arabinogalactan oligosaccharide has been obtained by enzymatic degradation, separation and purification, the sample with weight average molecular weight of 4500Da can obviously promote the growth of oligosaccharide AG2, thus it can be used as a probiotic food additive for promoting the growth of probiotics AG, such as well as a probiotic AG2 and a probiotic microorganism growth promoter.

The change of the parameters in the preparation method does not influence the preparation of the arabino-galacto-oligosaccharide, so the preparation of the arabino-galacto-oligosaccharide can be realized by the combination of any parameter in the preparation method. And will not be described in detail herein.

Claims

1, kinds of arabino-galacto-oligosaccharides, which is characterized by consisting of polysaccharide with the weight percentage of more than 99%, wherein the polysaccharide consists of arabinose and galactose, the molar ratio of the arabinose to the galactose is 1:16, and the weight average molecular weight of the arabino-galacto-oligosaccharides is 4000Da-5000 Da.

2. The arabinogalactan of claim 1, wherein the arabinose is α -arabinose and the galactose is β -galactose.

3. The arabinogalactan of claim 1, wherein the arabinose is α -L-arabinose and the galactose is β -D-galactose.

4. The arabinogalactan of claim 1, 2 or 3, wherein the structural units of the polysaccharide have a backbone of (1 → 3) linked β -D-galactose residues substituted at position 6 of the two β -D-galactose residues adjacent to the backbone by a branch and a terminal α -arabinose, respectively, and wherein the branch is a (1 → 6) linked β -D-galactose residue and a terminal β -D-galactose.

5. The method for producing arabinogalactan according to any one of claims 1 to 4 to , comprising the steps of:

(1) degrading, namely uniformly mixing 0.05mol/L citric acid buffer solution with β -1, 3-galactose incision enzyme and arabinogalactan, adjusting the pH value, performing enzymolysis for times, removing precipitate to obtain supernatant, continuously adding α -arabinosidase into the supernatant, adjusting the pH value, performing enzymolysis for the second time, and removing the precipitate to obtain degradation solution;

(3) and (3) drying: concentrating and drying the separated liquid obtained in the step (2) to obtain the arabino-galacto-oligosaccharide.

6. The method according to claim 5, wherein in the step (1), the β -1, 3-galactose oxidase is used in an amount of 10U to 45U: 1 g;

the usage amount of the α -arabinosidase and the arabinogalactan is 20U-65U: 1 g.

7. The preparation method according to claim 5, wherein in the step (1), the pH value of times of enzymolysis is 4.5-7.0, the enzymolysis temperature is 35-55 ℃, and the enzymolysis time is 30-60 h;

the pH value of the secondary enzymolysis is 5.5-7.5, the enzymolysis temperature is 45-65 ℃, and the enzymolysis time is 30-60 h.

8. The preparation method according to claim 5, wherein in the step (2), the ultrafiltration membrane has a molecular weight cut-off of 3000Da to 10000 Da;

the molecular weight cut-off of the nanofiltration membrane is 200Da-1000 Da.

9. The preparation method according to claim 5, wherein in the step (2), the parameters of membrane separation and purification by using the ultrafiltration membrane and the nanofiltration membrane are as follows: the pressure difference is 0.1MPa-0.5MPa, the temperature of the degradation liquid is 15-50 ℃, and the membrane area is 1.5m²-5m²Flux of degraded liquid film is 0.1m²/h-2.5m²/h。

10. Use of arabinogalactan according to any one of claims 1 to 4 to as an antioxidant, in the preparation of an antioxidant, as a probiotic growth promoter or in the preparation of a probiotic growth promoter.